Polypeptide for inhibiting nAChRs, product and application

Abstract

The embodiment of the invention relates to the technical field of polypeptides, and discloses a polypeptide for inhibiting nAChRs, a product and application. The polypeptide provided by the invention can generate a remarkable inhibition effect on nicotinic acetylcholine receptor mediated acetylcholine induced peak current amplitude, especially aiming at alpha 1 beta 1 epsilon delta, alpha 1 beta 1 delta gamma, alpha 7, alpha 9 alpha 10, alpha 3 beta 2 and alpha 4 beta 2 nAChR subtypes. Therefore, the polypeptide has important significance in the fields of medicine, makeup care, health care products and the like.

Description

Technical Field

[0001] This application relates to the technical field of peptides that inhibit nAChRs, and more particularly to a peptide, product, and application that inhibits nAChRs. Background Technology

[0002] Nicotinic acetylcholine receptors (nAChRs) are a class of ion channel receptors that play a crucial role in human physiological processes. They are widely distributed in many important physiological systems, including the central nervous system, peripheral nervous system, cardiovascular system, and immune system. Through their specific binding to acetylcholine (ACh), they participate in and regulate a variety of physiological functions.

[0003] In the nervous system, nAChRs ensure the accurate transmission of nerve signals to muscle cells at the neuromuscular junction, maintaining normal muscle contraction. Within the central nervous system, they are deeply involved in higher neural activities such as learning and memory, and play a crucial role in the progression of neurodegenerative diseases; their activation also possesses neuroprotective functions. In the cardiovascular system, nAChRs participate in heart rate regulation and vasodilation, influencing cardiac rhythm and blood pressure regulation. In the immune system, nAChRs are essential for regulating immune cell function and controlling inflammatory responses, playing a significant role in maintaining immune homeostasis. Furthermore, nAChRs are closely related to addictive behaviors, particularly in tobacco addiction, where nicotine binds to nAChRs, triggering dopamine release and leading to addiction.

[0004] The amplitude of the peak current induced by acetylcholine (Ach) has a profound impact on the function of nAChRs and related physiological and pathological processes. In the nervous system, abnormally elevated amplitudes can lead to neuronal overexcitation, triggering conditions such as epilepsy. In the cardiovascular system, abnormal changes can interfere with normal heart rhythm and vasomotor function, increasing the risk of cardiovascular disease. In the immune system, inappropriate amplitudes can disrupt the balance of immune regulation, leading to uncontrolled inflammatory responses. In the field of addiction, the amplitude of Ach-induced peak current is directly related to the reward effect of nicotine addiction; inhibiting this amplitude can effectively weaken dopamine release under nicotine stimulation, reducing addictiveness. When nAChRs function is abnormal, it may lead to an imbalance in the amplitude of Ach-induced peak current, which in turn affects signal transduction within skin cells, reducing collagen and elastin synthesis, resulting in signs of aging such as sagging skin and wrinkles. Furthermore, problems with nAChRs in other physiological systems can directly or indirectly affect a person's physical appearance and overall health.

[0005] Therefore, a peptide that can inhibit nAChRs is of great significance in the fields of medical and beauty care technologies. Summary of the Invention

[0006] To address the aforementioned problems, the first aspect of this application aims to provide a polypeptide that inhibits nAChRs, which is significant in inhibiting the peak current amplitude induced by acetylcholine (ACh) through human nicotinic acetylcholine receptors (nAChRs), and has important implications for the medical and cosmetic fields. The second aspect of this application aims to provide a product containing the polypeptide that inhibits nAChRs.

[0007] The third aspect of this application aims to provide an application of a product containing a polypeptide that inhibits nAChRs.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, there is a polypeptide that inhibits nAChRs, the polypeptide having an amino acid sequence as shown in SEQ ID NO.1, wherein the disulfide bonds of two cysteine ​​residues of the polypeptide are cyclic, and the C-terminus of the polypeptide is amidated.

[0010] Secondly, a polypeptide that inhibits nAChRs is provided for use in the preparation of medicines for the prevention, diagnosis or treatment of diseases caused by nAChR abnormalities, or for use in the preparation of medicines that indirectly achieve the prevention, treatment or diagnosis of other health problems by inhibiting nAChR activity.

[0011] Preferably, the nAChRs include α1β1εδ, α1β1δγ, α7, α9α10, α3β2 and α4β2 nAChR subtypes.

[0012] Preferably, the medicines for preventing, diagnosing, or treating diseases caused by nAChRs abnormalities include: medicines for treating muscle spasms, dystonia, tinnitus, cochlear damage, hypertension, gastrointestinal motility disorders, excessive sweating, chronic neuropathic pain, attention deficit / hyperactivity disorder, depression, anxiety, cognitive impairment, inflammation, tumor proliferation, epilepsy and excessive nerve excitability, neurogenic pruritus, and redness.

[0013] The drugs that indirectly prevent, treat, or diagnose other health problems by inhibiting nAChRs activity include anesthetic adjuvants and muscle relaxants used to block muscle nAChRs activity, antidotes for activating muscle nAChRs, and smoking cessation drugs used to treat nicotine dependence.

[0014] Thirdly, a peptide that inhibits nAChRs is provided for use in the preparation of beauty and personal care products for muscle relaxation, muscle spasms, mood regulation, skin anti-aging and wrinkle reduction, moisturizing and firming skin, or for the preparation of health products for improving immunity, enhancing concentration, improving ear health, anti-stress, and mood regulation.

[0015] Preferably, the mechanism of action of the beauty and personal care products and the health products lies in regulating the activity of α1β1εδ, α1β1δγ, α7, α9α10, α3β2 and α4β2nAChR subtypes.

[0016] Fourthly, a product for inhibiting nAChRs is provided, including the aforementioned peptides, including but not limited to pharmaceuticals, beauty and personal care products, health supplements, or scientific research reagents.

[0017] Preferably, the products include facial soothing patches and massage creams for antispasmodic relaxation; tinnitus relief patches and ear care essences for relieving ear discomfort; and aqueous solutions, emulsions, oils, creams, powders, and films for anti-wrinkle and anti-aging purposes.

[0018] Understandably, products can include excipients in addition to peptides. For example, when the product is a tablet, excipients may include fillers such as starch or other pharmaceutically acceptable excipients. When the product is an injectable drug or other liquid product, excipients may include solvents, buffers, solubilizers, chelating agents, and other excipients that do not affect the structure or properties of the peptide. In addition, excipients can also be the basic components in other dosage forms that do not affect the structure or properties of the peptide.

[0019] Meanwhile, the product dosage form can be any one of the following: cream, lotion, liquid, capsule, tablet, oil, powder, aerosol, or patch, such as face cream, lotion, serum, mask, spray, etc.

[0020] Specifically, α1β1δγ / α1β1εδ belong to muscle-type nAChRs, which are mainly distributed in the motor endplates of skeletal muscles and are key pathways for muscle contraction. The γ subunit is mainly found in fetal or denervated muscles, while the ε subunit replaces γ in adult skeletal muscle. Therefore, the peptides provided by this invention can act as inhibitors and can be applied to the following products or fields: as anesthetic adjuvants / muscle relaxants to block muscle nAChRs, suitable for surgical anesthesia; treatment of muscle spasms and dystonia; as antidotes for poisoning, such as against snake venom or some pesticide toxicity, the mechanism of action being the activation of muscle nAChRs leading to respiratory paralysis; as cosmetic and personal care products for local muscle relaxation to achieve cosmetic effects; or as antispasmodic and relaxing care products, including facial soothing patches, massage creams, etc.

[0021] α9 and α10 are mainly distributed in the hair cells of the inner ear and are used to regulate the amplification and feedback of auditory signals. Therefore, the polypeptides provided by this invention can be applied to the following products or fields: pharmaceuticals for the treatment of tinnitus by reducing abnormal auditory feedback signals; pharmaceuticals for hearing protection / noise damage protection; pharmaceuticals for the prevention of chemotherapy-related hearing damage; and functional ear care products, such as tinnitus relief patches and ear care serums.

[0022] α3β2 is mainly distributed in the pre- and post-ganglionic synapses of autonomic nerve ganglia, regulating sympathetic / parasympathetic nerve activity. Therefore, the polypeptide provided by this invention can act as an inhibitor and can be applied to the following products or fields: hypertension regulators; medicines for treating gastrointestinal motility disorders and excessive sweating; medicines for pain regulation; anti-stress health products; and skin care products related to regulating autonomic nervous system disorders, including skin care products for regulating flushing and stress acne.

[0023] α4β2 is mainly distributed in the central nervous system, especially in reward circuits, cognitive and attention regulation areas, and is the brain nAChR subtype with the highest expression level. Therefore, the peptide provided by this invention is suitable as an inhibitor and agonist in the pharmaceutical field, and can be applied to the following products or fields: similar to Varenicline, as a partial agonist in smoking cessation medications; used in medications to treat attention deficit hyperactivity disorder (ADHD), depression, anxiety, cognitive impairment, especially mild cognitive impairment; as a modulator of cognitive impairment such as Alzheimer's disease; and as a pain-modulating drug by regulating the release of dopamine and norepinephrine. In addition, in the consumer field, this peptide can also be used in energizing / focus-enhancing nutritional supplements or mood-regulating skin care products.

[0024] α7 is mainly distributed in the central nervous system, such as the cortex, hippocampus, and thalamus, participating in learning and memory, or in peripheral tissues, such as immune cells (macrophages, dendritic cells), intestines, and lungs. Therefore, the polypeptide provided by this invention is suitable as both an inhibitor and agonist in the pharmaceutical field, and can be applied to the following products or areas: immunomodulatory drugs, especially those acting on the cholinergic anti-inflammatory pathway to inhibit inflammation or enhance immune responses, such as drugs for the treatment of chronic hepatitis B; it can also be used as an adjuvant therapy for cancer, acting as an antagonist to reduce tumor proliferation caused by the overexpression of α7 in breast and lung cancer, thus achieving an anti-tumor effect; as a drug targeting epilepsy and excessive neural excitability, achieving sedation and anticonvulsancy by reducing neuronal firing; and as a drug or research reagent for research on targets in mental illnesses, such as for schizophrenia, where it can be used as an agonist control to verify target selectivity. In addition, peptides can also be used in immune-boosting health products, especially oral products, to enhance antiviral / anti-inflammatory responses, particularly in sub-healthy or viral infection contexts; or in antipruritic / analgesic skin care products, as an antagonist to relieve discomfort, especially neurogenic itching and redness-related symptoms caused by α7; or in anti-tumor related skin care products, such as functional serums containing α7 antagonistic peptides / plant extracts.

[0025] Based on the above-mentioned technical solution, the present invention has the following technical effects:

[0026] This invention provides a polypeptide that inhibits nAChRs. Experiments have demonstrated that this polypeptide significantly inhibits the amplitude of acetylcholine-induced peak current mediated by human (h) α1β1εδ, α1β1δγ, α7, α9α10, α3β2, and α4β2 nicotinic acetylcholine receptors. Furthermore, this polypeptide has been validated through cytotoxicity and membrane-penetrating ability experiments. Therefore, this polypeptide plays a crucial role and is of paramount importance in developing products targeting health problems or symptoms directly or indirectly caused by nAChR abnormalities. Attached Figure Description

[0027] Figure 1 The chromatogram of MetaSX is shown, with an elution time of 3.931 min.

[0028] Figure 2 The mass spectrum of MetaSX is shown, and the measured molecular weight is 2519.1, which is consistent with the theoretical value.

[0029] Figure 3 The study demonstrated the inhibitory effect of different concentrations of MetaSX on the amplitude of acetylcholine-induced peak current mediated by human (h) α1β1εδ, α1β1δγ, α3β2, α3β4, α4β2, α4β4, α7 and α9α10 nicotinic acetylcholine receptors.

[0030] Figure 4 The effect of different concentrations of MetaSX on the survival rate of HEK 293T cells.

[0031] Figure 5 The effect of different concentrations of MetaSX on the survival rate of human skin fibroblasts (BJ).

[0032] Figure 6 Experimental images to verify the membrane-penetrating ability of the peptide shown in SEQ ID NO.1 in H520 cells; where (a) is an image of H520 cells labeled with SX-FITC obtained by ordinary electron microscopy; and (b) is a fluorescence image of H520 cells labeled with SX-FITC obtained by fluorescence microscopy.

[0033] Figure 7 MetaSX was shown to promote the secretion of type I collagen from human skin fibroblasts (BJ).

[0034] Figure 8 MetaSX was shown to promote the production of type I and type III collagen in human skin fibroblasts (HFF-1); among which, Figure 8(a) Western blot images of type I and type III collagen in the control group and MetaSX group (concentration 80ppm / 40ppm) under the experimental conditions; Figure 8 (b) A statistical chart showing the semi-quantitative analysis of type I and type III collagen in the experimental group (concentration 80ppm / 40ppm) compared with the blank group and the MetaSX group.

[0035] Figure 9 MetaSX was shown to promote the expression of type I and type III collagen in a whole-skin organ-on-a-chip model; among which Figure 9 (a1) is an immunofluorescence staining image of the effect of MetaSX treatment on type I collagen expression. Figure 9 (a2) is Figure 9 Quantitative statistical graph of fluorescence intensity of (a1); Figure 9 (b1) is an immunofluorescence staining image of the effect of MetaSX treatment on type I collagen expression. Figure 9 (b2) is Figure 9 (b1) Quantitative statistical graph of fluorescence intensity.

[0036] Figure 10 This demonstrates that FITC-MetaSX can effectively penetrate the dermis of a full-skin organ-on-a-chip model.

[0037] Figure 11 for Figure 10 A magnified view of a portion of the image.

[0038] Figure 12 The study showed that MetaSX improved skin stratum corneum hydration and transepidermal water loss at 30 min, 14 d, and 28 d after administration; among which... Figure 12 (a1) Statistical graph of skin stratum corneum moisture content at different time points after MetaSX was used in the subjects and in the blank group; Figure 12 (a2) is a statistical graph showing the epidermal water loss values ​​of subjects after using MetaSX and the control group at different time points.

[0039] Figure 13 Skin radiance was measured in subjects at 30 min, 14 d, and 28 d after MetaSX application.

[0040] Figure 14 The study showed that MetaSX improved skin elasticity and firmness in subjects at 30 minutes, 14 days, and 28 days after application; among which... Figure 14 (a1) Statistical graph of skin elasticity test at different time points for subjects after using MetaSX and for the blank group; Figure 14 (a2) is a statistical graph showing the results of the test on tightness in subjects after using MetaSX and in the blank group at different time points.

[0041] Figure 15 The study showed that MetaSX improved skin roughness and smoothness at 30 min, 14 d, and 28 d in the subjects; among which Figure 15 (a1) Statistical graph of skin roughness at different time points after the subjects used MetaSX and the blank group; Figure 15 (a2) is a statistical graph showing the skin smoothness of subjects after using MetaSX and the blank group at different times.

[0042] Figure 16 Chin angle values ​​for subjects at 14d and 28d after using MetaSX.

[0043] Figure 17 Changes in crow's feet wrinkles in subjects at 14 and 28 days after using MetaSX; among them Figure 17 (a1) Statistical chart of the change in the proportion of crow's feet area after MetaSX processing; Figure 17 (a2) Images of skin taken for MetaSX processing to show changes in crow's feet wrinkles.

[0044] Figure 18 Changes in under-eye wrinkles in subjects 14 and 28 days after using MetaSX; Figure 18 (a1) Statistical chart of the change in the proportion of under-eye wrinkles after MetaSX processing; Figure 18 (a2) Images of skin taken for MetaSX processing to show changes in under-eye wrinkles.

[0045] Figure 19 Changes in nasolabial folds in subjects 14 and 28 days after using MetaSX; Figure 19 (a1) Statistical chart of the change in the proportion of nasolabial fold area after MetaSX processing; Figure 19 (a2) Images of skin taken for MetaSX processing to show changes in nasolabial folds. Detailed Implementation

[0046] To facilitate understanding of the present invention, a more comprehensive description will be provided below with reference to specific embodiments. Preferred embodiments are given herein. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0047] Before further describing in more detail the various embodiments of the compounds / compositions and methods of this disclosure through exemplary descriptions, examples, and results, it should be understood that the embodiments of this disclosure are not limited in application to the details of the methods and compositions described below. The descriptions provided herein are for illustrative purposes only and are not intended to be interpreted in a limiting sense. The inventive concept of this disclosure can have other embodiments or can be practiced or implemented in various ways. Therefore, the language used herein is intended to give the broadest scope and meaning; and the embodiments are intended to be exemplary, not exhaustive, and are not intended to limit this disclosure to these particular embodiments. Moreover, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting unless otherwise stated. Furthermore, numerous specific details are set forth in the following detailed description to provide a more thorough understanding of this disclosure.

[0048] However, it will be apparent to those skilled in the art that embodiments of this disclosure can be practiced without these specific details. In other instances, features well-known to those skilled in the art have not been described in detail to avoid unnecessary complexity. It is intended that all substitutions, replacements, modifications, and equivalents that are apparent to those skilled in the art are included within the scope of this disclosure. Based on this disclosure, all compounds / compositions disclosed herein, their preparation methods, applications, and uses can be prepared and implemented without excessive experimentation.

[0049] Therefore, although the compounds / compositions and methods of this disclosure have been described with reference to specific embodiments, it will be apparent to those skilled in the art that variations may be made to the formulations, compounds or compositions and / or methods, as well as the steps or sequence of steps of the methods described herein, without departing from the spirit and scope of the inventive concept of this disclosure.

[0050] As used herein, any reference to "an embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. The phrase "in an embodiment" appearing in multiple places throughout the specification does not necessarily refer to the same embodiment.

[0051] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0052] Example 1: Artificial Synthesis of a Peptide that Inhibits nAChRs

[0053] The specific preparation steps are as follows:

[0054] (1) Solid-phase synthesis

[0055] The Fmoc solid-phase synthesis method was employed, using Rink Amide resin to achieve C-terminal amidation of the peptide chain. Following the target sequence, corresponding Fmoc-protected amino acids were sequentially added from the C-terminus to the N-terminus. The coupling reagent used was HBTU / HOBt, the base was DIEA, and the solvent was DMF. After each coupling reaction, Fmoc deprotection was performed using 20% ​​piperidine / DMF. In this embodiment, the target amino acid sequence is shown in SEQ ID NO.1, specifically GYLACRPRRMRHRKRMLAFC, where a disulfide bond is formed between Cys5 and Cys20, and the C-terminus of the peptide is amidated.

[0056] (2) Cutting and removing protection

[0057] After peptide synthesis, the aforementioned resin was placed in a mixture of trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) / ethylene dithiol (EDT) (volume ratio 94:2.5:2.5:1) and reacted at room temperature for 2 hours to cleave the peptide chain from the resin and remove the side-chain protecting groups. After the reaction was complete, the resulting reaction solution was added to cold anhydrous diethyl ether for precipitation, and the crude peptide was obtained by centrifugation.

[0058] (3) Oxidation and cyclization

[0059] The crude peptide was dissolved in 0.1M ammonium bicarbonate buffer (pH 8.0) to maintain a concentration of 0.1–0.2 mg / mL. The solution was stirred at room temperature for 24 hours, or allowed to stand for 24 hours, to allow Cys5 and Cys20 to spontaneously oxidize and form disulfide bonds, achieving head-to-tail cyclization. After the cyclization reaction was complete, the peptide was freeze-dried to obtain the crude cyclic peptide.

[0060] (4) Purification and identification

[0061] Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) with a C18 column and a gradient elution system of 0.1% TFA in water and acetonitrile in the mobile phase. Specifically, the HPLC analysis conditions for the sample were as follows: a Phenomenex-C18(2)luna3um 100A 4.6*100mm A2117# column was used; a mobile phase consisting of two solutions was used, where phase A was an aqueous solution of 0.1% TFA (trifluoroacetic acid); phase B was a mixed solution of 80% ACN (acetonitrile) and 20% water containing 0.09% TFA; the flow rate was set to 1.0 ml / min; and gradient elution was performed by linearly increasing the proportion of phase B from 27.0% to 37.0% within 10 minutes. The molecular weight of the pure product was confirmed by mass spectrometry, and the formation of disulfide bonds was determined. The specific results are attached. Figure 1 and attached Figure 2 As shown.

[0062] After purification and identification, the purity of the target product obtained in this preparation process is greater than 95%. The target product in this embodiment is named MetaSX, that is, the polypeptide that can inhibit nAChRs obtained in this embodiment will be referred to as MetaSX below.

[0063] Example 2 Electrophysiological Activity Test

[0064] A human nAChRs expression system was constructed in Xenopus laevis oocytes using a dual-electrode voltage clamp technique and microinjection method for electrophysiological activity assay.

[0065] The specific preparation steps are as follows:

[0066] (1) nAChRs cRNA acquisition

[0067] First, we need to ensure that the required human nAChRs gene has a suitable vector containing the T3 promoter, and then linearize the plasmid using a restriction endonuclease (such as NotI). Next, we use T3 RNA polymerase for in vitro transcription to generate capped cRNA. The cRNA is then purified using LiCl precipitation, and its concentration and purity are measured at A260 / A280 wavelengths. The cRNA is then aliquoted and stored at -80°C. The entire process must be strictly enzyme-free to prevent cRNA degradation and inactivation.

[0068] (2) African Xenopus egg retrieval procedure

[0069] One day before the procedure, feed the frog a small amount of food. After egg retrieval, due to surgical wound pain and limited mobility, the African clawed frog will not eat for about a week. Select mature African clawed frogs and anesthetize them for 30 minutes using an ice-water mixture to induce hibernation. During anesthesia, ensure the frog is upright, with more ice than water, burying it in ice, ensuring the head is inside the ice. Collagenase is diluted to 2 mg / mL with 1*OR2 (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl, 5 mM HEPES, pH 7.4) for subsequent cell processing. During the surgery, bury the frog's head and limbs in ice. 2-3 cm above the pubic bone on the abdomen, use surgical scissors to remove the abdominal skin and muscle tissue. Use forceps to remove the eggs through the incision and cut off the egg sac. If the retrieved eggs cannot be processed immediately, they can be stored in OR-2 solution at 4°C. After the frog eggs are removed, the wounds of the African clawed frog are sutured using absorbable sutures. Three to four stitches are used to suture the muscle tissue, with three surgical knots on each side to prevent bleeding after the frog awakens. The same procedure is performed on the outer skin. After processing, the African clawed frog is placed belly-up in a pool along with an ice-water mixture to await awakening. Under a microscope, the removed frog egg tissue is initially separated using forceps, tearing the vascular membrane into small pieces without damaging the egg cells. Then, using a flipper, the cells are placed in a 2 mg / mL collagenase solution for digestion for 20 minutes. The condition of the frog eggs should be monitored frequently during this time to prevent over-digestion. After digestion, wash three times with OR-2 solution, discard any damaged cells floating on the surface, and then wash three times with ND-96 solution (96.0mM NaCl, 2.0mM KCl, 1.8mM CaCl2, 1.0mM MgCl2, 5mM HEPES, pH 7.5). Incubate at 4°C.

[0070] (3) nAChRs cRNA microinjection

[0071] When introducing target nAChR cRNA into oocyte cytoplasm using microinjection, injection parameters must be precisely controlled according to the characteristics of the receptor subtype. The hα7nAChR subtype requires 10 ng of cRNA, the hα9α10nAChR subtype requires 35 ng of cRNA, while other subtypes use a standard injection dose of 5 ng. This dosage difference is based on the expression efficiency and functional characteristics of different receptor subtypes on the oocyte membrane surface. After injection, cells should be immediately transferred to ND-96 culture medium containing antibiotics and incubated statically in a 16°C sterile incubator. The entire expression cycle typically lasts 48-72 hours. During this period, ion channel function should be monitored using a voltage-clamp system. Successful functional expression of the receptor protein is considered achieved when the transmembrane current amplitude stabilizes and the signal-to-noise ratio meets experimental standards. In this embodiment, the target subtype nAChRs include α1β1εδ, α1β1δγ, α3β2, α3β4, α4β2, α4β4, α7 and α9α10.

[0072] (4) Electrophysiological recording

[0073] After the dual-electrode voltage clamp system was debugged, oocytes stably expressing the target protein were placed in a 30 μL microrecording cell. Using a micromanipulator, two glass microelectrodes (filled with 3 mol / mL KCl electrode solution) were precisely inserted into the cell membrane, and a stable seal was established by setting the clamping voltage to -80 mV. Throughout the experiment, the cellular physiological environment was maintained using a gravity perfusion system (flow rate 2 mL / min): under normal conditions, ND-96 extracellular solution (containing 96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.0 mM HEPES, pH 7.4) was used; for cells expressing hα9α10nAChRs, a high-sodium ND-115 extracellular solution (115.0 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.4) was used. After the cell membrane potential stabilized and the baseline noise level remained below 20 nA, three stimulations with the same concentration of ACh were administered. Each stimulation was followed by a 3-minute rinse interval to ensure receptor desensitization and recovery. The average of the three induced current peaks was taken as the baseline current value (I0). Subsequently, the test solution containing the candidate peptide (the target product prepared in Example 1) was replaced for perfusion. After the recording chamber was completely replaced (approximately 1.5 minutes), flow was stopped, and the chamber was incubated for 5 minutes to allow the peptide to fully bind to the receptor. After removing the peptide solution, ACh stimulation was repeated to obtain the post-inhibition current value (I1). Peptide activity was expressed as the current inhibition rate, calculated using the formula: (I0-I1) / I0×100%. Each concentration gradient was independently repeated three times. The entire experiment used an Axoclamp 900A amplifier to acquire the raw signals. After baseline correction, noise filtering, and peak detection were performed on the raw data using Clampfit 10.2 software, dose-response curve fitting and statistical analysis were performed using GraphPad Prism 8. One-way ANOVA was used for intergroup comparisons, and the data are expressed as mean ± SEM.

[0074] The results are as follows Figure 3 As shown, Figure 3 The given bar charts show the inhibitory effects of MetaSX (1 and 10 μM) on the amplitude of acetylcholine (ACh)-induced peak currents mediated by human (h) α1β1εδ, α1β1γδ, α3β2, α3β4, α4β2, α4β4, α7, and α9α10 nicotinic acetylcholine receptors (nAChRs). Whole-cell currents of hα1β1εδ and hα1β1γδ were activated by 5 μMACh; hα3β2, hα4β4, and hα9α10 were activated by 6 μMACh, respectively; and hα3β4, hα4β2, and hα7 were activated by 300 μM, 3 μM, and 100 μMACh, respectively (mean ± standard deviation, n = 6–11).

[0075] MetaSX can effectively inhibit nAChR isoforms α1β1εδ, α1β1δγ, α7, and α9α10 at a concentration of 1 μM; it can effectively inhibit α1β1εδ, α1β1δγ, α7, α9α10, α3β2, and α4β2 at a concentration of 10 μM.

[0076] Based on the above, the half-inhibition concentration IC was also calculated. 50 And nH (Hill coefficient), the specific values ​​are shown in Table 1 below.

[0077] Table 1. Blocking activity (half-blocking dose, IC50) and nH (Hill coefficient) of MetaSX against different acetylcholine receptor subtypes.

[0078]

[0079] These results indicate that MetaSX has a high inhibitory effect and positive synergistic effect on the α1β1εδ, α1β1δγ, α7, α9α10, α3β2, and α4β2 subtypes, especially the α1β1εδ, α1β1δγ, α7, and α9α10 subtypes, which is of great significance for drug development, disease diagnosis, and other fields.

[0080] Example 3 Cytotoxicity Test

[0081] To assess the biocompatibility of MetaSX, we used HEK 293T and human skin fibroblast (BJ, Cell Bank of Chinese Academy of Sciences, GNHu49) models for cell viability experiments. After counting cells in the logarithmic growth phase, we resuspended and pelleted them in complete culture medium. Based on cell size and growth rate, the cell suspension was evenly seeded into 96-well plates (100 μL per well, approximately 5000-10000 cells). Due to the edge effect of the 96-well plate, which can cause evaporation of the surrounding medium, we only seeded the central 60 wells, adding 200 μL of sterile PBS to the remaining peripheral wells. Cells were incubated at 37°C in a 5% CO2 incubator for 12-36 hours to allow for full cell adhesion. The peptides were dissolved in sterile pure water to prepare a high-concentration stock solution, which was then serially diluted with complete culture medium to twice the target final concentration. A blank control group (200 μL culture medium), a negative control group (100 μL cells, 100 μL culture medium), and experimental groups (100 μL cells, 100 μL peptide solutions diluted with complete culture medium at different concentrations) were set up, and cultured for 24-48 h. Under light-protected conditions, 20 μL of CCK-8 reagent (10% of the original culture medium volume) was added directly to each well, gently vortexed horizontally to mix, and incubated at 37°C for 4 h in the dark. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. If air bubbles or precipitates were present in the wells, centrifugation at 1000 rpm for 5 minutes was performed before reading to eliminate interference. Each concentration was tested in triplicate. Cell viability (%) was calculated as (OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group) * 100%.

[0082] The results are as follows Figure 4 and Figure 5 As shown, even at a high concentration of 100 μM, the survival rate of HEK 293T cells was not affected by MetaSX. Similarly, even at a high concentration of 60 ppm, the survival rate of BJ cells was not affected. These results indicate that MetaSX is non-toxic to cells.

[0083] Example 4: Membrane penetration test

[0084] MetaSX cells were N-terminally labeled with FITC (SX-FITC). The labeling process introduced a flexible linker. H520 cells were cultured and grown in 1640 medium containing 10% FBS at 37°C and 5% CO2. When cell confluence was approximately 80%, the cells were detached using trypsin. For the SX-FITC labeling experiment, 8 x 10⁵ cells were seeded into 12-well dishes and cultured to approximately 80% confluence overnight. The next day, the growth medium was removed, and the cells were washed twice with PBS. Then, the cells were incubated for 4 hours in 1640 medium containing SX-FITC (30 μM). The cells were washed twice with PBS to remove unbound SX-FITC, and the SX-FITC-labeled cells were then observed and photographed using a fluorescence microscope.

[0085] The results are as follows Figure 6 As shown, MetaSX can enter H520 cells, indicating that MetaSX has good cell penetration ability.

[0086] Example 5: In vitro collagen-promoting activity test

[0087] To verify the collagen-promoting effect of MetaSX in this invention, a series of in vitro cell and whole skin organ-on-a-chip experiments were conducted on MetaSX.

[0088] Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using GraphPad Prism 9.0 software, with significance thresholds defined as: ***P < 0.001, **P < 0.01, *P < 0.05.

[0089] (1) Promotes collagen secretion from human skin fibroblasts (BJ).

[0090] Human skin fibroblasts (BJ, Cell Bank of Chinese Academy of Sciences, GNHu49) were collected at a concentration of 2 × 10⁻⁶. 4 Seeds were seeded per well in 96-well plates and cultured overnight to 80% confluence. A blank control group (with added culture medium), a positive control group (with added medium containing 40 ppm nAChR inhibitory peptide Syn-ake), and an experimental group (with added medium containing 10 / 20 ppm MetaSX) were set up. Incubation was performed at 37°C for 48 h, with three biological replicates per group. The supernatant was collected and the secreted type I collagen content was detected using an ELISA kit (ab210966, abcam).

[0091] The results are as follows Figure 7 As shown, the addition of 10 / 20 ppm MetaSX significantly promoted the secretion of type I collagen in BJ cells.

[0092] (2) Promotes collagen production in human skin fibroblasts (HFF-1)

[0093] Human skin fibroblasts (HFF-1, Cell Bank of Chinese Academy of Sciences, SCSP-109) were harvested at a concentration of 2 × 10⁻⁶ cells / cell. 5 Cells were seeded per well in 6-well plates and cultured overnight to 70% confluence. A blank control group (with added culture medium) and an experimental group (with added medium containing 40 / 80 ppm MetaSX) were set up and incubated at 37°C for 48 h. Each group had 3 biological replicates. The supernatant was discarded, and the cells were washed twice with PBS. Cell lysis buffer was added, and the cells were lysed on ice to collect proteins. The content of type I and type III collagen was detected by Western blotting. Antibody information is as follows: Collagen Type I antibody (67288-1-Ig, Proteintech, Wuhan Sanying Biotechnology Co., Ltd.); Collagen Type III antibody (68320-1-Ig, Proteintech, Wuhan Sanying Biotechnology Co., Ltd.). Using β-tubulin as a reference protein, protein bands were semi-quantitatively analyzed using ImageJ software.

[0094] The results are as follows Figure 8 As shown, adding 40 ppm MetaSX significantly promotes the production of type III collagen in HFF-1 cells, while adding 80 ppm MetaSX significantly promotes the production of both type I and type III collagen in HFF-1 cells.

[0095] (3) Promote collagen expression in whole skin organ microarray model

[0096] A full-skin organ-on-a-chip model was constructed. A blank control group (supplemented with culture medium) and an experimental group (stimulated with culture medium containing 10 ppm MetaSX for 60 min, then washed three times with pre-warmed PBS) were set up, repeated for 5 days, for a total stimulation time of 300 min. 24 h after the last stimulation, the cells were fixed with 4% paraformaldehyde for 2 h. Type I and type III collagen were labeled by immunofluorescence staining, and fluorescence distribution was observed and collected using a laser confocal microscope (NIKONAX). Antibody information was collected as above. Fluorescence intensity was analyzed using ImageJ software.

[0097] The results are as follows Figure 9 As shown, adding 10 ppm MetaSX significantly enhanced the expression of type I and type III collagen in the entire skin model.

[0098] These results suggest that MetaSX can help remodel the skin's extracellular matrix (ECM) and combat skin aging.

[0099] Example 6: Permeability Test

[0100] To verify the skin permeability of MetaSX of the present invention, in vitro experiments were conducted on MetaSX.

[0101] MetaSX was covalently labeled with FITC in 0.1M sodium carbonate buffer (pH≈9) to prepare a 1 mg / mL fluorescent peptide solution. The solution was diluted to 10 ppm, and 200 μL was added to the surface of a whole-skin organ-on-a-chip model and incubated at a constant temperature for 2 h. The model was washed twice with PBS, fixed with 4% paraformaldehyde for 6 h, and embedded in OCT at -20℃ using a 10% / 20% / 30% sucrose gradient. 20 μm frozen sections were then excised. The fluorescence distribution at different skin depths was observed and acquired using a NIKONAX laser confocal microscope.

[0102] The results are as follows Figure 10 and Figure 11 As shown, after 2 hours of treatment with 10 ppm MetaSX, MetaSX effectively penetrated into the epidermis and upper dermis of the whole-skin organ-on-a-chip model. The results indicate that MetaSX has a sustained and effective penetration potential into the skin.

[0103] Example 7: Human trials of MetaSX efficacy

[0104] To verify the efficacy of MetaSX on various indicators of human facial skin, 42 subjects were randomly selected, and 33 were included. Two subjects withdrew from the test midway due to personal reasons, resulting in valid data from 31 subjects. All subjects were female, with a mean age of 48.1 years. A double-blind, half-face randomized controlled trial design was used, dividing subjects into two groups. Baseline (D0) skin parameters of both cheeks were recorded. The control group applied serum B (15ppm acetyl hexapeptide-8) to both cheeks after cleansing in the morning and evening, while the experimental group applied the test serum (15ppm MetaSX). Both groups used the serum continuously for 28 days.

[0105] Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using GraphPad Prism 9.0 software. The significance thresholds were defined as follows: compared to baseline (D0), ***P < 0.001, **P < 0.01, *P < 0.05; compared to the control group, ###P < 0.001, ##P < 0.01, #P < 0.05.

[0106] (1) Moisturizing efficacy test

[0107] After using the sample essence for 30 minutes, 14 days, and 28 days, the stratum corneum moisture content of the skin on both cheeks was measured using a Corneometer CM825 skin moisture meter, and the transepidermal water loss (TEWL) value of both cheeks was measured using a Tewameter™ He skin water loss meter.

[0108] The results are as follows Figure 12 As shown in (a1), the skin stratum corneum moisture content of the experimental group was significantly higher than that of the control group using acetyl hexapeptide-8 after 30 min, 14 d, and 28 d of MetaSX treatment, indicating higher skin stratum corneum moisture content in the experimental group. Figure 12 As shown in (a2), the TEWL values ​​after using MetaSX for 30 min, 14 d, and 28 d were significantly lower than those in the control group, indicating that the experimental group had less transdermal water loss per unit time and per unit cross-sectional area. These results demonstrate that MetaSX has superior barrier moisturizing properties for the skin.

[0109] (2) Brightening effect test

[0110] After using the sample essence for 30 minutes, 14 days, and 28 days, the skin luster of the left and right cheeks was measured using a Glossymeter GL200 skin luster testing probe.

[0111] The results are as follows Figure 13 As shown, the skin radiance of subjects in the experimental group was significantly higher than that of the control group using acetyl hexapeptide-8 after 30 minutes, 14 days, and 28 days. The results indicate that MetaSX can increase skin radiance and brighten skin tone.

[0112] (3) Elasticity and firming effect test

[0113] After using the sample essence for 30 minutes, 14 days, and 28 days, the subjects measured the skin elasticity R2 value and skin firmness parameter F4 value of both cheeks using a CutometerMPA 580 skin elasticity tester.

[0114] The results are as follows Figure 14 As shown in (a1), the skin elasticity R2 values ​​of the subjects in the experimental group were significantly higher than those of the control group using acetyl hexapeptide-8 after 30 min, 14 d, and 28 d, indicating better skin elasticity in the experimental group. Figure 14 As shown in (a2), the skin firmness F4 values ​​after 30 min, 14 d, and 28 d of MetaSX treatment were significantly lower than those in the control group, indicating stronger skin firmness in the experimental group. These results suggest that MetaSX can increase skin elasticity and firmness, and reduce skin sagging.

[0115] (4) Smoothing effect test

[0116] After using the sample essence for 30 minutes, 14 days, and 28 days, the subjects measured the skin roughness (SEr) and skin smoothness (SEsm) values ​​of both cheeks using the VisioScanVC20 plus skin surface texture analysis system.

[0117] The results are as follows Figure 15 (a1) and Figure 15 As shown in (a2), the experimental group showed significantly better improvement in skin roughness and smoothness after using MetaSX for 28 days compared to the control group using acetyl hexapeptide-8. These results indicate that consistent use of MetaSX can improve rough skin and make it smoother.

[0118] (5) Test on the effectiveness of improving chin contour

[0119] After 14 and 28 days of using the sample essence, the subjects had images of the skin on both cheeks acquired using the VISIA CR image analysis system, and the mandibular angle value was obtained using IPP analysis.

[0120] The results are as follows Figure 16 As shown, the experimental group showed a significantly greater reduction in chin angle after using MetaSX for 28 days compared to the control group using acetyl hexapeptide-8. The results indicate that consistent use of MetaSX can improve chin contour.

[0121] (6) Anti-aging efficacy test

[0122] After 14 and 28 days of use of the sample essence, the subjects had their skin images of both cheeks collected using the VISIA CR image analysis system. IPP analysis was used to obtain the area ratio of crow's feet, under-eye wrinkles, and nasolabial folds.

[0123] The results are as follows Figure 17-19 As shown, after 28 days of MetaSX use, the experimental group showed significantly lower proportions of crow's feet, under-eye wrinkles, and nasolabial folds compared to the control group using acetyl hexapeptide-8. These results indicate that MetaSX can reduce crow's feet, under-eye wrinkles, and nasolabial folds, demonstrating excellent anti-wrinkle and anti-aging effects.

[0124] The above description is merely an example and illustration of the structure of this invention, and while the description is specific and detailed, it should not be construed as limiting the scope of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this invention, and these obvious substitutions all fall within the protection scope of this invention.

Claims

1. A polypeptide that inhibits nAChRs, characterized in that, The polypeptide has an amino acid sequence as shown in SEQ ID NO.1, wherein the disulfide bonds of the two cysteine ​​residues of the polypeptide are cyclic, and the C-terminus of the polypeptide is amidated.

2. The use of a polypeptide that inhibits nAChRs in the preparation of a medicine for the prevention, diagnosis or treatment of diseases caused by nAChR abnormalities, or in a medicine for the indirect prevention, treatment or diagnosis of other health problems by inhibiting nAChR activity.

3. The application of the polypeptide that inhibits nAChRs according to claim 2 in the preparation of pharmaceutical products for the prevention, diagnosis, or treatment of diseases caused by nAChR abnormalities, or in pharmaceutical products for the indirect prevention, treatment, or diagnosis of other health problems by inhibiting nAChR activity, characterized in that... The nAChRs include α1β1εδ, α1β1δγ, α7, α9α10, α3β2 and α4β2 nAChR subtypes.

4. The application of the polypeptide that inhibits nAChRs according to claim 3 in the preparation of pharmaceutical products for the prevention, diagnosis, or treatment of diseases caused by nAChR abnormalities, or in the preparation of pharmaceutical products that indirectly achieve the prevention, treatment, or diagnosis of other health problems by inhibiting nAChR activity, characterized in that... The medicines mentioned for the prevention, diagnosis or treatment of diseases caused by nAChRs abnormalities include: medicines for muscle spasms, dystonia, tinnitus, cochlear damage, hypertension, gastrointestinal motility disorders, excessive sweating, chronic neuropathic pain, stress, attention deficit / hyperactivity disorder, depression, anxiety, cognitive impairment, immune regulation, tumor proliferation, epilepsy and excessive nerve excitability, neurogenic pruritus, and redness. The drugs that indirectly prevent, treat, or diagnose other health problems by inhibiting nAChRs activity include anesthetic adjuvants and muscle relaxants used to block muscle nAChRs activity, antidotes for activating muscle nAChRs, and smoking cessation drugs used to treat nicotine dependence.

5. The application of a peptide that inhibits nAChRs in the preparation of beauty and personal care products for muscle relaxation, muscle spasms, mood regulation, skin anti-aging and wrinkle reduction, moisturizing and firming, and skin brightening, or in the preparation of health products for improving immunity, enhancing concentration, improving ear health, anti-stress, and mood regulation.

6. The application of a polypeptide that inhibits nAChRs in the preparation of beauty and personal care products for muscle relaxation, muscle spasms, mood regulation, skin anti-aging and wrinkle reduction, hydration and moisturizing, and firming and brightening of the skin, or in the preparation of health products for improving immunity, enhancing concentration, improving ear health, anti-stress, and mood regulation, characterized in that, The mechanism of action of the beauty and personal care products and the health products lies in regulating the activity of α1β1εδ, α1β1δγ, α7, α9α10, α3β2 and α4β2nAChR.

7. A product for inhibiting nAChRs, characterized in that, Includes the polypeptide described in claim 1.

8. The product according to claim 7, characterized in that, This includes facial soothing patches and massage creams for antispasmodic relaxation; tinnitus relief patches and ear care serums for relieving ear discomfort; and aqueous solutions, emulsions, oils, creams, powders, and patches for anti-wrinkle, anti-aging, hydrating, or firming and brightening skin.

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